Endocannabinoids inhibit leptin function in the hypothalamus 1 Negative Regulation of Leptin-induced ROS Formation by CB 1 Receptor Activation in Hypothalamic Neurons Letizia Palomba 1,2 , Cristoforo Silvestri 2,3# , Roberta Imperatore 2,3 , Giovanna Morello 2,3 , Fabiana Piscitelli 2,3 , Andrea Martella 2,3 , Luigia Cristino 2,3 and Vincenzo Di Marzo 2,3* 1 Department of Biomolecular Sciences, University of Urbino “Carlo Bo”, Urbino, Italy. 2 Endocannabinoid Research Group; 3 Institute of Biomolecular Chemistry, Consiglio Nazionale delle Ricerche, Pozzuoli, Italy; Running title: Endocannabinoids inhibit leptin function in the hypothalamus * To whom correspondence may be addressed: Istituto di Chimica Biomolecolare, Consiglio Nazionale delle Ricerche, Via Campi Flegrei 34, Comprensorio Olivetti, 80078, Pozzuoli (NA), Italy. Tel.: +39-081- 8675018. Fax: +39-081-8041770. E-mail: vdimarzo@icb.cnr.it Keywords: Leptin; endocannabinoid receptor 1; ROS; endocannabinoids; hypothalamus. Background: In hypothalamic neurons, leptin induces ROS production via PPAR-Ȗ inhibition. Results: CB1 agonism prevents leptin-induced ROS accumulation by reversing PPAR-Ȗ and catalase inhibition. Inhibition of endocannabinoid inactivation also counteracts leptin effects. Conclusion: CB 1 inhibits effects of leptin that underlie part of its anorexic actions. Significance: During conditions of increased endocannabinoid tone, CB 1 might reduce leptin activity in the hypothalamus. ABSTRACT The adipocyte-derived, anorectic hormone, leptin, was recently shown to owe part of its regulatory effects on appetite-regulating hypothalamic neuropeptides to the elevation of ROS levels in arcuate nucleus (ARC) neurons. Leptin is also known to exert a negative regulation on hypothalamic endocannabinoid levels and hence on cannabinoid CB 1 receptor activity. Here we investigated the possibility of a negative regulation by CB 1 receptor of leptin- mediated ROS formation in the ARC. Through pharmacological and molecular biology experiments we report data showing that leptin-induced ROS accumulation is: 1) blunted by arachidonyl-2'-chloroethylamide (ACEA) in a CB 1 -dependent manner, in both the mouse hypothalamic cell line mHypoE-N41 and ARC neuron primary cultures; 2) likewise blocked by a peroxisome proliferator-activated receptor-γ (PPAR-γ) agonist, troglitazone, in a manner inhibited by T0070907, a PPAR-γ antagonist, which also inhibited ACEA effect on leptin; 3) blunted under conditions of increased endocannabinoid tone due to either pharmacological or genetic inhibition of endocannabinoid degradation in mHypoE-N41 and primary ARC neuronal cultures from MAGL -/- mice, respectively; 4) associated with reduction of both PPAR-γ and catalase activity, which are reversed by both ACEA and troglitazone. We conclude that CB 1 activation reverses leptin-induced ROS formation, and hence possibly some of the ROS-mediated effects of the hormone, by preventing PPAR-γ inhibition by leptin, with subsequent increase of catalase activity. This mechanism might underlie in part CB1 orexigenic actions under physiopathological conditions accompanied by elevated hypothalamic endocannabinoid levels. In the central nervous system, the hypothalamus, originally recognized as the brain “feeding center”, is the main regulator of body weight. Hormonal and nutrient signals are processed in this brain area and inform the rest of the brain and the body about the free and stored levels of fuel available for the organism (1). In turn, hypothalamic neuronal circuits use this information to regulate caloric intake, energy consumption, and peripheral lipid and glucose metabolism. Among the chemical mediators involved in this regulation, the endocannabinoids (ECs) are master controllers of the fast (i.e., non- genomic) and stress-related fine-tuning of energy intake and processing. The endocannabinoid http://www.jbc.org/cgi/doi/10.1074/jbc.M115.646885 The latest version is at JBC Papers in Press. Published on April 13, 2015 as Manuscript M115.646885 Copyright 2015 by The American Society for Biochemistry and Molecular Biology, Inc. by guest on April 20, 2015 http://www.jbc.org/ Downloaded from